We propose to reveal some central aspects of the molecular basis of metabolic and insulin regulation of glycogen metabolism and glucose homeostasis. This is to be done by focusing on the key enzyme glycogen phosphorylase. The molecular details of the regulation principles are to be investigated by a combination of single-crystal X-ray kinetics using purified liver and muscle phosphorylases and their interconverting enzymes and also using a viable rat hepatocyte cell system. We shall determine an accurate set of atomic coordinates at 2.0 A resolution for muscle phosphorylase a in the inhibited (T) conformation. Using these coordinates and the Difference Fourier Technique, numerous ligand binding studies will be done at high resolution to accurately define the structure of the several distinct effector sites and possibly the active site as they interact with various metabolites. We shall initiate an X-ray structural study on crystals which have the enzyme in its active (R) conformation. The structural and kinetic model as proposed from the muscle enzyme system will be tested functionally by in vitro kinetic studies of the metabolite regulation of liver-enzyme and its interconversion by phosphatase and kinase. The in vivo metabolic and insulin regulation of glycogen phosphorylase will be studied using viable rat hepatocytes to test the models derived from the X-ray and in vitro kinetic studies.